Abstract Hematopoietic cell transplantation (HCT) is a potentially curative therapy for a broad range of hematological malignancies and other disorders, however, acute GVHD is a major complication that limits the use and results in significant morbidity and mortality.1-3 About half of patients with acute GVHD will have steroid refractory disease that carries an especially high mortality with few treatment options. Because there are currently no clinical tests to prospectively identify or treat steroid refractory GVHD, therapy is largely empiric.2 My central long-term goal is to combine the use and development of T cell immune monitoring and T cell based immunotherapy to improve outcomes for HCT patients and, eventually, many other patients. In this application, we propose work that could redefine how we understand, diagnose and treat GVHD with implications for many other immunologic diseases. We propose to apply cutting-edge nucleotide sequencing- based approaches to identify and track T cells causing GVHD. We propose to show that this knowledge can be used to guide the development and implementation of improved monitoring and new therapies to treat these patients. This includes the development of immunosuppressive Tregulatory cells to treat GVHD We propose to study pre-clinical models of GVHD and to test if novel 'educated' donor CD25+ Tregs that appear to treat active and severe GVHD might be translated to the clinic. This will be accomplished if the phenotype of eTregs is clarified and we have answered the question as to whether or not these cells impair graft-versus tumor response and function in clinically relevant settings. We anticipate that eTregs will require MHC interactions to be generated, depend less upon IL-2 to function and will prevent the expansion of GVHD- associated T cell clones identified by T cell repertoire sequencing and/or response to known GVHD antigens. We propose also to defining T cell signatures that predict, confirm or stratify acute GVHD in patients. We propose to integrate flow cytometry and single cell genomics into this approach, advances that could finally allow clinicians to accession the function of the adaptive immune system in making clinical decisions. We will accomplish the goals of our study if we confirm TCR sequencing can be used to risk stratify steroid refractory patients or extended to help predict GVHD. In addition, the aim will be accomplished if we can identify the phenotype and treatment response of T cells highly implicated in GVHD, both for patients undergoing conventional HCT and for those receiving Tregs for the prevention or treatment of GVHD. As an extension of this work, we have conceived of novel approaches to identify GVHD target antigens by integrating high- throughput TCR sequencing for GVHD target antigen discovery. This proposal will allow scientists and clinical practitioners to better understand and control alloimmunity and hopefully help to prevent or cure GVHD.